This invention is directed to a bioassay for determining the functionality of parathyroid hormone compounds. More particularly, this invention is directed to a bioassay wherein the compound to be tested is added to a culture of parathyroid hormone receptor expressing cells bearing a reporter gene under the transcriptional control of multiple c-AMP responsive elements. Still more particularly, this invention is directed to a bioassay wherein the compound to be tested is added to a culture of parathyroid hormone receptor expressing cells bearing a luciferase reporter gene under the transcriptional control of multiple c-AMP responsive elements.
Human parathyroid hormone (hPTH) is an 84 amino acid protein which is a major regulator of calcium homeostasis. Parathyroid hormone-related protein (hPTHrP) is a 139 to 171 amino acid protein with N-terminal homology to hPTH. The N-terminal fragments of hPTH and hPTHrP, particularly those consisting of amino acids 1-34, retain the full biological activity of the parent hormone.
The biological activity of hPTH is reflected in the activation of two secondary messenger systems: G-protein coupled adenylyl cyclase (AC) and protein kinase C (PKC) activity. The N-terminal fragments hPTH(1-34)OH and hPTH(1-31)NH2 have been demonstrated to be anabolic with respect to bone formation in humans and ovariectomized rats, respectively. This increase in bone growth has been demonstrated to be coupled with stimulation of adenylyl cyclase activity. Analogs of these N-terminal fragments have significant therapeutic potential for the treatment of physiological conditions associated with bone cell calcium regulation including hypocalcemia; osteoporosis; osteopenia; and disorders associated with osteoporosis and osteopenia such as hyperparathyroidism, hypoparathyroidism, and Cushings syndrome; glucocorticoid- and immunosuppressant-induced osteopaenia; and bone fracture and bone refracture repair.
To facilitate the discovery of efficacious hPTH analogs, a need exists for methods of determining the functionality of these analogs. Such a method should be simple, sensitive, and lend itself to automation so that a multiplicity of compounds can be rapidly screened.
This invention is directed to a bioassay for determining the functionality of a parathyroid hormone compound comprising
(a) adding the compound to a culture of parathyroid hormone receptor expressing cells bearing a reporter gene under the transcriptional control of multiple cAMP responsive elements; and
(b) measuring the change in expression of the reporter gene. Preferably, the change in reporter gene expression is compared to appropriate control levels of reporter gene expression. Appropriate controls which may be measured include, but are not limited to, expression in the absence of the parathyroid hormone compound and/or expression in the presence of a known parathyroid hormone receptor agonist. Parathyroid hormone receptor agonists include, but are not limited to, hPTH(1-34)OH and hPTH(1-31 )NH2.
The cells may be prokaryotic or eukaryotic cells. Preferably, the cells are mammalian cells. The cells may comprise an endogenous or heterologous nucleic acid encoding the parathyroid hormone receptor. Preferably, the cells comprise a heterologous nucleic acid encoding the parathyroid hormone receptor.
The number of cAMP responsive elements should be sufficient to drive expression within the cell of the reporter gene in the presence of cAMP. Preferably, the number of cAMP responsive elements is greater than about 10. More preferably, the number of cAMP responsive elements is about 16.
The reporter gene may encode a protein selected from, but not limited to, xcex2-galactosidase, chloramphenicol acetyltransferase, xcex2-glucuronidase, and luciferase. A preferred reporter gene encodes luciferase.